Leucovorin and its salts are known to be pharmaceutically effective. See, Remington's Pharmaceutical Services, Mack Publishing Co., Easton, PA 1985 (Remington's) p. 1023. Kerwar et al., U.S. Pat. No. 4,746,662 disclose that the antiarthritic efficacy of methotrexate can be potentiated by injection of an aqueous solution of leucovorin or its salts. EPO Patent Publication No. 0,266,042, May 4, 1988, describes using pure leucovorin isomers to manufacture medicaments for methotrexate rescue, for treatment of colorectal cancer in combination with 5-fluorouracil, and for treating folate deficiency. In U.S. Pat. No. 4,500,711, Wisowaty et al., describe the purification of leucovorin and its salts.
Leucovorin is normally administered in the form of salts such as alkaline metal and alkaline earth metal salts, such as the calcium salt of leucovorin, with the 1-isomer being preferred.
The compound N-(((2-amino-5-formyl-3,4,5,6,7,8-hexahydro-4-oxo-6-pteridinyl)methyl)amin o)benzoyl)-L-glutamic acid, calcium salt (1:1), pentahydrate, (Leucovorin Calcium USP) is sold commercially as the calcium salt of a 1:1 mixture of formulae (Ia and Ib) for which the compounds have the (R) and (S) stereo-chemistry respectively at C-6. ##STR1## It is used principally as an antidote for folic acid antagonists such as methotrexate, which blocks the conversion of dihydrofolic acid to tetrahydrofolic acid. Leucovorin salts are formulated in water for injection with suitable preservatives, as described under Leucovorin Calcium Injection in the Physician's Desk Reference, Forty-third Edition, Medical Economics Company, Oradell, NJ 1989 (PDR 43rd Ed.) p. 1124.
The pharmacokinetic behavior of the two isomers differs in that the (S)-isomer IIb) is selectively absorbed from the gastrointestinal tract and has a shorter plasma half-life relative to the (R)-isomer (Ia).
The naturally occurring isomer Ib, which is the 6S diastereoisomer, has been reported (C. Temple, Jr., J. P. Rose, W. R. Laster, and J. A. Montgomery, Cancer Treatment Reports, 65, 1117-1119 (1981)) to be important for rescue therapy by virtue of its effectiveness at restoring one-carbon metabolism.
A report (R. P. Leary, Y. Gaumont, and R. L. Kisliuk, Biochem. and Biophys. Res. Commun., 56, 484-488 (1974)) that thymidylate synthesis from L. casei is inhibited by the non-natural diastereoisomer of 5,10methylene tetrahydrofolate and a report (V. F. Scott and K. O. Donaldson, Biochem. and Biophys. Res. Commun., 14, 523-526 (1964)) that 5,10-methylene tetrahydrofolate dehydrogenase from E. coli is also inhibited by the same diastereoisomer coupled with the observation (G. K. Smith, P. A. Benkovic, and S. J. Benkovic, Biochem., 20, 4034-4036 (1981)) that the same diastereoisomer of 10-formyltetrahydrofolate is a potent competitive inhibitor of Glycinamide ribonucleotide formyltransferase (GAR) from chicken liver points to the inhibition of both pyrimidine and purine biosynthesis and thus of DNA biosynthesis by the non-natural diastereoisomers of one-carbon derivatives of tetrahydrofolate. Therefore, the non-natural forms cannot be considered as biologically inert. If such inhibition is present in mammalian systems, then a potential clinical requirement for only the natural (6S) form of tetrahydrofolates, especially leucovorin, exists.
The diastereoisomeric components Ia and Ib have been separated (D. B. Cosulich, J. M. Smith, Jr. and H. P. Proglist, J. Am. Chem. Soc., 74, 4215 (1953)) by fractional crystallization and by chromatography (J. Feeney, B. Birdsall, J. P. Albrand, G. C. K. Roberts, A. S. Bungen, P. A. Charlton and D. W. Young, Biochem., 20, 1837 (1981)). A stereospecific reduction of dihydrofolate catalyzed by dihydrofolate reductase has been reported (L. Rees, E. Valente, C. J. Suckling and H. C. S. Wood, Tetrahedron, 42, 117 (1986)) to afford the 6(S)-isomer stereospecifically.
A paper by L. Rees, E. Valente, C. Suckling and H. C. S. Wood, Tetrahedron, 42, 117-136 (1986) describes the synthesis of chiral derivatives of tetrahydrofolate, including leucovorin. This system required the use of dihydrofolate reductase from E. coli and an extensive and expensive recycling of reducing cofactor NADPH. Another paper by L. Rees, C. J. Suckling and H. C. S. Wood, J. Chem. Soc. Chem. Commun. 470, (1987) (European Patent Application 0 266 042 A2) describes the acylation of 6(R,S)-tetrahydrofolate with (-)-menthyl chloroformate to give N-5 derivatives as a diastereoisomeric mixture which were separated by fractional precipitation. Subsequent treatment of each diastereoisomer with formic acid and hydrogen bromide in acetic acid followed by hydrolysis gave each diastereoisomer of 5-formyltetrahydrofolate.
It has now been found that the optically pure diastereoisomers of tetrahydrofolate compounds can be produced easier in accordance with this invention. This process has a major advantage over previously published or patented procedures in that it is surprisingly simple, because the key step requires only the use of one enzyme, tetrahydrofolate formylase, otherwise called formate activating enzyme or formyltetrahydrofolate synthetase (FTHFS), which also selectively adds the required formyl group to only the 6S diastereoisomer when using a racemic mixture of 5,6(R,S),7,8-tetra-hydrofolic acid (IIa,b). Further, the utilization of this enzyme has a distinct advantage over the use of the enzyme dihydrofolate reductase in that an extensive regeneration system for cofactors does not have to be employed.
Tetrahydrofolate formylase can be elaborated by Micrococcus aerogenes, Clostridium cylindrosporum, Clostridium acidi-urici, Clostridium thermoaceticum, and by other microorganisms, plants and animals. D. H. Butlaire, Methods In Enzymology, 66, 585-599 -(1980).
By using radiolabeled ammonium formate or any other, including in situ made, radiolabeled formic acid salts or derivatives in the enzymatic formylation, labeled (IIIb) can be obtained and converted to labeled (IVb) or labeled (Ib). With the aid of the enzyme 5,10-methylenetetrahydrofolate dehydrogenase any of the radiolabeled, formyl group carrying 5,6S,7,8-tetrahydro-folates (Ib,IIIb, or IVb), preferably (IVb), can be transformed to labeled 5,10-methylene-5,6S,7,8-tetrahydro-folic acid which in turn, under the action of the enzyme 5,10-methylenetetrahydrofolate reductase can be converted to radiolabeled 5-methyl-5,6S,7,8-tetrahydrofolic acid. Thus, radiolabeled (Ib) generated by the application of this invention is a useful compound for the production of radiolabeled 5,6S,7,B-tetrahydrofolic acid derivatives.
The invented process also unexpectedly allows for the isolation of 5,6R,7,8-tetrahydrofolic acid (IIa) which is not formylated by the enzyme. Isolated (IIa) can then be sold as such (commercialized as a rare compound) or serve as the starting material for the production of, for example, 5-formyl-5,6R,7,8-tetrahydrofolic acid (Ia) in an analogy to a procedure described by R. G. Moran and P. D. Colman, Anal. Biochem. 122, 70-78 (1982) using formic acid with or without the water-soluble carbodiimide, 1-ethyl-3-(3 -dimethyl-aminopropyl) carbodiimide. Preferable, however, is the isolation of (IIa) after the conversion of (IIIb) to (IVb) and/or (Ib) has been completed. This allows for an easier separation of the components by reverse-phase chromatography, for example. Also, (IIa) can be modified in the presence of (IVb) and/or (Ib) , since the 5-position of (IIa) is still reactive whereas that of (IVb) or (Ib) is not, to produce derivatized (IIa) , for example 5-carboxymenthyl-5,6R,7,8-tetrahydrofolic acid, which can be separated from (IVb) or (Ib) in a simple adsorption step.
Furthermore, separated (IIa) can be reacted as reported by C. Temple, Jr. , L. L. Bennett, Jr. , J. D. Rose, R. D. Elliott, J. A. Montgomery and J. H. Mangum, J. Med. Chem., 25, 161-166 (1982) to prepare various 5- and 10- substituted, 5,10-disubstituted , and 5,10-bridgesubstituted 5,6R,7,8-tetrahydrofolic acid derivatives. In particular, 5-methyl-5,6R,7,8-tetrahydrofolate can be synthesized from (IIa) with formaldehyde and sodium borohydride under basic conditions as described in a method by J. A. Blair and K. J. Saunders, Anal. Biochem. , 34, 376 (1970). Alkylation of (IIa) with dimethylsulfate in N,N-dimethylacetamide at 55.degree. C. is achieved by using a method described in Japanese patent 73 32,120 (1973); Chem. Abst. , 80, 2792X (1974) .
By substituting formic acid or the alkylating agents in the above indicated reactions with radiolabeled formic acid or derivatives thereof or radioactive alkylating agents, radiolabeled 5,6R,7,8-tetrahydrofolic acid derivatives will be obtained.
in accordance with this invention, both radio-labeled 5,6S,7,8-tetrahydrofolic acid derivatives and radiolabeled 5,6R,7,8-tetrahydrofolic acid derivatives and their salts can be produced separately, which are useful compounds for testing, for example studying enzymatic mechanisms.